United States consumers spend more than $4 billion each year on fresh market tomatoes. During the summer months, most of these fresh market tomatoes are grown on farms located throughout the United States and then sold locally. During the cooler months, when locally grown tomatoes are not available, most of these tomatoes are grown in the southern portions of the United States and in Mexico and then shipped by truck throughout the rest of the country. Unfortunately, when these southern grown tomatoes are allowed to fully ripen on the vine before shipping, they do not remain in marketable condition long enough for supermarkets to shelve them and for consumers to buy them.
To prevent the tomatoes from rotting before they reach consumers, farmers typically pick, pack, and ship the tomatoes while green. Before sale, the green tomatoes are gassed with ethylene to redden them. These unripened “gassed” tomatoes do not spoil quickly, but they have developed a reputation for poor flavor, especially compared to the summer “vine-ripened” tomatoes.
Due to consumer dissatisfaction with the unripened “gassed” tomatoes, research and breeding efforts have focused on developing tomatoes that exhibit a longer shelf-life when they are allowed to ripen fully on the vine. One approach to developing longer shelf-life tomatoes is to use traditional breeding techniques, i.e., crossing tomato plants with desired characteristics and selecting those progeny plants with fruits exhibiting longer shelf-lives. While traditional breeding techniques have been used to develop most of the tomato cultivars used by growers today, these methods are very time intensive. It can take years to breed a novel tomato variety that may exhibit only a modest increase in shelf life.
Another approach to developing longer shelf-life tomatoes is to use genetic techniques to manipulate the biochemical and physiological changes associated with the ripening process in tomatoes. One biochemical change in ripening fruit is the depolymerization and solubilization of cell wall polyuronides by the ripening-induced cell wall degrading enzyme, polygalacturonase (PG). Tomato fruit PG (Della Penna et al., Proc. Natl. Acad. Sci. U.S.A. 1986 83:6420-6424; Bird et al., Plant Mol. Biol. 1988 11:651-662) belongs to a family of tomato PG genes. PG enzyme activity increases dramatically during the ripening of many fruits, including tomato, and is the primary enzymatic activity responsible for cell wall polyuronide degradation.
For example, in U.S. Pat. Nos. 5,107,065; 5,442,052; 5,453,566; 5,569,831; and 5,759,829, tomato plants were transformed with DNA constructs encoding an antisense oligonucleotide for the PG gene. When expressed, the foreign DNA provided an RNA sequence capable of binding to the naturally existing mRNAs of the PG gene in the transformed tomato plant thereby preventing the translation of the mRNA into the PG protein. The fruit of transformed tomato plants showed improved properties in terms of slower softening post harvest, thereby increasing the shelf-life of the tomato.
Another research group, using a complicated series of transgenic manipulations involving transposon sequences from another plant species, created a “knock out” of the PG gene in tomato. Enzymatic analysis of fruit from plants containing the knock out of the PG gene showed at least a 1000-fold reduction in PG levels. See Cooley, M. B. and Yoder, J. I., Plant Mol. Biol., 1998 Nov. 1, 38(4):521-30; Cooley et al., Mot. Gen. Genet. 1996 Aug. 27, 252(1-2):184-194.
This anti-sense and “knock-out” work indicates that fruit PG gene expression is not necessary for viable, normal tomato fruit production. While several features of the ripening process remain normal, transgenic tomatoes having reduced PG gene expression exhibit slower softening post harvest and increased shelf life. Additionally, these transgenic tomatoes exhibit a lower incidence of post-harvest disease infection due to the preservation of intact fruit skin and coat caused by the delayed softening. Therefore, the tomatoes with reduced PG have fewer cosmetic blemishes which deter customers.
Reduced PG enzyme activity is important not only to the fresh market tomato industry but also to the processed tomato industry. During commercial processing of tomatoes, pectin integrity of the tomato is lost by enzymatic degradation of the pectin by PG. In order to avoid this degradation, a rapid, high heat treatment is used to destroy the PG enzyme activity. The annual cost associated with the total energy required to bring millions of tons of tomatoes to a temperature sufficient to rapidly inactivate the PG enzyme is a significant cost to the tomato processing industries.
While the use of these genetic techniques has resulted in producing tomatoes with reduced PG gene expression, the genetic techniques used to date employ recombinant DNA being introduced into tomatoes. Since many consumers have clear preferences against genetically modified foods, it would be useful to have a tomato exhibiting reduced levels of fruit PG that was not the result of genetic engineering methods. However, to date, no one has ever found or described a naturally occurring “knockout” of the endogenous tomato PG gene. Therefore, a tomato with its fruit PG gene either knocked out or otherwise hindered would have tremendous value to the entire tomato industry.